A change in the structure Or conformation of prion protein (PrP) may be responsible for the development of prion diseases, a group of neurodegenerative disorders. Such hypothesis is supported by the fact that mutations in the open reading frame of human PrP gene have been linked to the familial forms of the diseases known as Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia. An essential element in the design of a rational therapy for prion diseases is the detailed knowledge of molecules that interact with PrP. Previous studies have indicated that PrP binds to sulfated glycans, some of which represents the glycosaminoglycan (GAG) moiety of proteoglycans. The present proposal will examine the molecular basis and pathological significance of PrP-GAG interaction. Two specific aims have been put forth to model such interaction in vitro. In Specific Aim 1, the GAG binding affinities of PrP carrying different mutations will be determined by heparin affinity coelectrophoresis. The structural motif of PrP responsible for GAG binding will be characterized by enzymatic digestion, ligand blotting, heparin affinity chromatography, and N-terminal sequence analysis. In Specific Aim 2,the effect of GAG binding on detergent solubility and cellular processing of both mutant and wild-type PrP will be examined in cells derived from patients with familial prion diseases, using pulse-chase paradigm, surface biotinylation, and immunoprecipitation. The proposed studies will likely lead to a better understanding of the importance of the GAG interaction in PrP metabolism with implications for the pathogenesis of prion diseases. In addition, the in vitro model may help design of effective therapeutics for the prevention and treatment of prion diseases.